Genetic Characterization of a Core Set of a Tropical Maize Race Tuxpeño for Further Use in Maize Improvement

Genetic Characterization of a Core Set of a TropicalMaize Race Tuxpeno for Further Use in MaizeImprovementWeiwei Wen1,2*., Jorge Franco3., Victor H. Chavez-Tovar2, Jianbing Yan1, Suketoshi Taba2*

1 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei, China, 2 International Maize and Wheat Improvement Center

(CIMMYT), El Batan, Mexico, 3 International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria

Abstract

The tropical maize race Tuxpeno is a well-known race of Mexican dent germplasm which has greatly contributed to thedevelopment of tropical and subtropical maize gene pools. In order to investigate how it could be exploited in future maizeimprovement, a panel of maize germplasm accessions was assembled and characterized using genome-wide SingleNucleotide Polymorphism (SNP) markers. This panel included 321 core accessions of Tuxpeno race from the InternationalMaize and Wheat Improvement Center (CIMMYT) germplasm bank collection, 94 CIMMYT maize lines (CMLs) and 54 U.S.Germplasm Enhancement of Maize (GEM) lines. The panel also included other diverse sources of reference germplasm: 14U.S. maize landrace accessions, 4 temperate inbred lines from the U.S. and China, and 11 CIMMYT populations (a total of 498entries with 795 plants). Clustering analyses (CA) based on Modified Rogers Distance (MRD) clearly partitioned all 498 entriesinto their corresponding groups. No sub clusters were observed within the Tuxpeno core set. Various breeding strategies forusing the Tuxpeno core set, based on grouping of the studied germplasm and genetic distance among them, werediscussed. In order to facilitate sampling diversity within the Tuxpeno core, a minicore subset of 64 Tuxpeno accessions(20% of its usual size) representing the diversity of the core set was developed, using an approach combining phenotypicand molecular data. Untapped diversity represents further use of the Tuxpeno landrace for maize improvement through thecore and/or minicore subset available to the maize community.

Citation: Wen W, Franco J, Chavez-Tovar VH, Yan J, Taba S (2012) Genetic Characterization of a Core Set of a Tropical Maize Race Tuxpeno for Further Use inMaize Improvement. PLoS ONE 7(3): e32626. doi:10.1371/journal.pone.0032626

Editor: Lewis Lukens, University of Guelph, Canada

Received August 7, 2011; Accepted January 30, 2012; Published March 7, 2012

Copyright: � 2012 Wen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The Government of Japan and the United States Department of Agriculture (USDA) supported this work. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected] (WW); [email protected] (ST)

. These authors contributed equally to this work.

Introduction

Knowledge of genetic diversity within and among maize

landraces is essential for effectively managing the conservation of

landraces and using them in plant breeding. Maize landraces have

genetic diversity in terms of plant and ear morphology, adaptation,

and consumer traits such as grain quality and yields. Following

studies based upon chromosomal knob morphology [1,2] and

isozyme markers [3–8], several analyses of maize landraces using

DNA markers have been carried out [9–12]. Based on genotyping

193 landrace accessions at 99 microsatellite loci, Matsuoka et al. [9]

presented phylogenetic analysis indicating a single domestication for

maize and developed a scenario for its spread through the Americas.

Reif et al. [10] used 25 simple sequence repeat (SSR) markers to

characterize 25 maize race accessions from Mexico and examined

their relationships on the basis of morphological data. Vigouroux et

al. [11] analyzed the population genetic structure of maize races by

genotyping 964 individual plants, representing most of the entire set

of about 350 races native to the Americas, with 96 microsatellites.

They identified the highland of Mexico and the Andes as potential

sources of genetic diversity, which are currently underrepresented

among elite lines in maize breeding programs. Most recently,

Sharma et al. [12] revealed significant phenotypic and microsatel-

lite-based genetic diversity in 48 landrace accessions in India, and

identified promising accessions which could be utilized for

introgression of novel traits in broad-based pools/populations.

The tropical maize race Tuxpeno has been incorporated in

pools and populations in CIMMYT [13], where pools are maize

populations with a broad genetic base. Its productivity per se and

combining ability in crossing with race ETO developed at

Estacion Tulio Ospina, Colombia is known as Tuxpeno-ETO

heterotic patterns in tropical maize breeding [14–16]. It is

predominantly a white dent with a cylindrical ear type. Some

accessions of race Tuxpeno are yellow dent type, which were

collected mainly in the Huasteca region of San Luis Potosi,

Hidalgo, and Veracruz in Mexico. The long-term accessions

evaluation experiments at CIMMYT planted 2,366 accessions of

the race Tuxpeno since 1988. From them, 1,350 accessions were

uniquely identified to be the race Tuxpeno. They are mostly from

Mexico, but also include introductions from Brazil, Ecuador,

Guatemala, and Venezuela. A multivariate cluster analysis of

phenotypic data collected from seven trials was used to create a

core set containing 321 accessions (23.7% of 1,350 Tuxpeno race

accessions) of the race Tuxpeno [17–24].

PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e32626

CIMMYT has developed and released CIMMYT maize lines

(CMLs) since 1984. The CMLs are carefully selected with good

general combining ability (GCA) and a significant number of

value-added traits such as drought tolerance, nitrogen use

efficiency, acid soil tolerance, and resistance to disease and insect

pests: (http://www.cimmyt.org/ru/component/content/article/

459-international-maize-improvement-network-imin/434-cimmyt-

maize-inbred-lines-cml). They are used as parental lines for the

hybrids in one to several maize mega-environments (MEs). Two

heterotic patterns were classified within CMLs (i.e. CML-A as dent

kernel type and CML-B as flint kernel type). CMLs were developed

from tropical, subtropical and highland white and yellow dent

CIMMYT populations and pools, including germplasm from

Central America, Caribbean, Mexico, South America, and USA.

Some of them originated from populations and gene pools with a

background of Tuxpeno germplasm.

The GEM project in the United States is designed to broaden

U.S. maize breeding germplasm, representing a public-private

sector collaboration in which elite tropical and sub-tropical

germplasm (i.e. from non-Corn Belt dent races of maize) is

crossed with private sector inbred lines (http://www.public.iastate.

edu/,usda-gem/). GEM has used some of the elite germplasm of

the Latin American Maize Project (LAMP) identified as a source

of new genetic diversity for broadening the genetic base of U.S.

maize hybrids, and breeding crosses are grouped into stiff stalk

(SS) and non-stiff stalk (NSS) heterotic patterns [25–28]. As

Tuxpeno germplasm has not been largely used in the GEM

project, comparison of genetic diversity of them would be of

interest to maize breeders.

In this study, the Tuxpeno core set containing 321 accessions,

together with 14 U.S. landrace accessions, 11 CIMMYT

populations, 4 temperate inbred lines, 94 CMLs and 54 GEM

lines was characterized using SNPs across the maize genome. The

objectives were to assess genetic diversity and genetic distance

among the Tuxpeno core and other germplasm; to investigate

potential utilization of the Tuxpeno core in maize improvement

and to develop a minicore subset of the Tuxpeno core to facilitate

sampling untapped alleles, if they existed.

Materials and Methods

Plant materials genotyped in this studyA total of 498 accessions were assembled in this study including

321 landrace accessions of Tuxpeno core set (two individual plants

each accession except 24 accessions with one plant investigated),

94 CMLs, 54 GEM lines, 4 temperate maize inbred lines (Mo17,

CI7_1, DAN340, K22_1), 14 landrace accessions from the U.S.,

and 11 CIMMYT populations (6 CIMMYT populations and 5

single cross hybrids between CMLs) (Table 1). Leaf samples of all

498 accessions (795 individual plants) were taken from individual

plants at seedling stage. DNA was extracted using a modified

CTAB procedure according to Murray et al. [29].

Within this Tuxpeno core, 295 accessions from Mexico, 22

accessions from Guatemala, 2 accessions from Brazil, and one

each from Ecuador and Venezuela were included. Thus, the

geographic origins of the Tuxpeno core are from Guatemala and

Chiapas-Nuevo Leon (east coast), Veracruz-Nayarit (central

region), and Colima-Sinaloa (west coast) of Mexico. The set of

94 CMLs includes lines from CIMMYT heterotic group A (n = 48)

and B (n = 38), and 8 lines of A/B pattern. These lines were first

chosen in seed production nurseries of CIMMYT maize

germplasm bank for well adapted lines in the CIMMYT tropical

and subtropical stations at Agua Fria and Tlaltizapan in cycle A,

2008. Thirty-five lines included in the U.S. GEM panel are stiff

stalk (SS) heterotic pattern and 19 lines are non-stiff stalk (NSS)

heterotic pattern. GEM SS lines included 25% germplasm of

tropical hybrids from Brazil, Mexico, and Thailand, and landraces

from Argentina, Brazil, and the Caribbean (Cuba), and 75% of

elite temperate germplasm. GEM NSS lines included 25%

germplasm of landraces from Brazil, Caribbean (Saint Croix),

Chile, Mexico, Uruguay, and a hybrid of DKXL370 (Brazil).

Within other germplasm, entries of ‘‘Across 8443’’, CML 247

(G.24)6CML 254 (P.21), population 21, population 43, CML 444

(P.43), and CML 445 (Tux. Sequilla) have a background of mainly

Tuxpeno germplasm. Pop. 28 and Pool 26 are yellow dent with

slight Tuxpeno germplasm. Pop.32 (ETO Blanco) and Pop.23

(Blanco Cristalino: Pool 23) are white flint populations. Hybrids

are included to represent those tolerant to drought, including lines

with Tuxpeno background. CML395 (IITA 90323), CML 202

(ZSR923), CML312SR (P.500+SR), CML442 (Recycled in

M37W/ZM607) have diverse origins. 14 U.S. landrace accessions

are southern dent and Corn Belt dent. Detailed information of

these lines collected and characterized in this study is listed in

Table S1.

Phenotypic evaluation and formation of Tuxpeno coreset

Seven trial sets mentioned above were conducted during 1988

to 2008 at three CIMMYT experimental stations (i.e., Tlaltizapan,

18u419480N, 99u079480W, 940 m above sea level; Agua Fria,

20u279000N; 97u 389 240W, 100 m above sea level; and Poza Rica,

20u 339 000N; 97u 279 000W, 60 m above sea level). The

experimental design used alpha lattice with two replications. Each

plot consisted of two 5 m rows with 75 cm apart between rows.

Two seeds per hill were sown and later thinned to establish 32

plants per plot. Six check entries were included in each trial at

each experiment station. Forty-four traits were evaluated for each

accession, including morphological (plant height; ear height; ratio

of ear height to plant height; tillering in scale; tassel type;

percentage of erect plants; grain type; grain color), agronomic

(days to 50% anthesis; days to 50% silking; ratio of anthesis to

silking; foliar disease scale; root lodging (%); stalk lodging (%);

number of plants harvested; number of ears harvested; ratio of

harvested ears to harvested plants; field ear weight per plot (kg);

rating on ear rot; rating on easiness of shelling; ear quality; grain

moisture (%); grain shelling (%); adaptation in scale; agronomic

scale; ratio of grain yield (kg) to grain moisture(%); yield per

hectare (kg/ha)), vegetative (germination (%); rating on seedling

vigor; number of leaves above the ear; days to leaf senescence;

ratio of days to silking to days to leaf senescence; rating on forage

production; rating on pubescence; rating on husk cover) and

reproductive traits (ear length; ear diameter; kernel length; kernel

width; kernel row number per ear; ratio of ear diameter to ear

length; cob diameter; ratio of cob diameter to ear diameter; ratio

of kernel width to kernel length). Detailed information of these

traits can be found in Table S2. A multivariate cluster analysis

(Ward-MLM) and a sample allocation strategy-D method and

selection indexes (ESIM), were used to select core set to represent

phenotypic diversity of the race Tuxpeno [17–23]. All trait data of

discrete and continuous variables (44 traits in total) were included

in calculating Gower distance among the accessions [24]. Based on

the Gower distance, Ward was used to make a preliminary

grouping, which was improved by MLM using maximum

likelihood estimation. For each accession in the core set, the

accession name, trial set in which they were evaluated, race

classification, the value of each trait in the separate trial sets and

the mega-environments (MEs) that they originated from are listed

in Table S2.

Genetic Characterization of Maize Race Tuxpeno


SNP genotypingGenotyping was performed using Illumina GoldenGate assay on

1,536 bi-allelic SNP markers developed by Yan et al. [30]. The

details of the SNP genotyping procedure and allele scoring have

also been described [30]. The software Illumina BeadStation 500

G (Illumina, Inc., San Diego, CA, USA) was used for SNP

genotyping according to the protocol described by Fan et al. [31].

Allele calling was re-checked manually and further analysis was

carried out.

Clustering analysis and genetic diversityA neighbor-joining tree of these 498 entries was constructed

based on the Modified Rogers genetic distance (MRD) using 1,041

SNPs. Briefly, pair-wise MRD between each two entries were

calculated using an R (http://www.R-project.org) code, and

neighbor-joining method implemented in the DARwin5 (http://

darwin.cirad.fr/darwin) program was used on the matrix of

distances to construct the dendrogram. An additional tree was

constructed to show the relationship among different germplasm

groups (Tuxpeno core, CML-A, CML-B, CML-A/B, GEM-SS,

GEM-NSS, CIMMYT populations, U.S. landraces), based on the

Nei’s genetic distance [32]. Bootstrap support for this tree was

determined by resampling across 1,041 SNP loci for 1000 times.

The output of each bootstrap sample was summarized to obtain a

consensus tree.

The genetic diversity parameters gene diversity and observed

heterozygosity were quantified for sets of entries. Gene diversity,

often referred to as expected heterozygosity, is defined as the

probability that two randomly chosen alleles from the population

are different. The estimator of gene diversity is defined for the rth

locus as Dr~1{Pm

i~0 �X 2i , where m is the number of alleles and

Xi is the population frequency of the ith allele at locus r [33].

Adaptation and genetic divergence of Tuxpeno coreA GIS–based approach for defining global maize production

environments called ‘‘mega-environments (MEs)’’ has been useful

for targeting maize germplasm for the introduction and adaptation

trials [34]. The program DIVA-GIS (http://www.diva-gis.org/)

was used to assign the maize growing environments based on the

altitude, latitude and longitude information of the accessions. The

MEs of 299 Tuxpeno accessions were defined based on their

available geographic information.

Within the Tuxpeno core, 277 accessions were classified into 10

subgroups according to the 10 major geographic regions (i.e.

Guatemala and 9 states in Mexico: Chiapas, Hidalgo, Jalisco,

Nayarit, Nuevo Leon, Sinaloa, San Luis Potosi, Tamauripas,

Veracruz) where they were collected from (Table 1), based on

available passport data. The program Arlequin [35] was used to

perform analysis of molecular variance (AMOVA; [35,36]) and

investigate the population differentiation among these 10 sub-

groups; and statistical significance of each variance component as

well as pair-wise Fst was assessed based on 1000 permutations of

the data.

Minicore subset formationData of 44 phenotypic traits (i.e. 31 continuous, 11 categorical

and two nominal variables; Table S2, [21]) and genotypic data

(1,433 SNPs covering 10 chromosomes) from evaluation of 321

Tuxpeno accessions were used to develop a minicore subset with a

sample size equal to 20% of the entire core set size (that is 64

accessions). Morphological Gower distance [24] and MRD [37]

were calculated between every pair of the 321 accessions and then

combined following the Gower principle of using the average of

both the two distances weighted by the number of variables

included in the distance calculations, where MRD accounted for

Table 1. Tuxpeno core and diverse germplasm used for genotyping.

Germplasm category Origins of germplasm (country or state in Mexico) Number of accessions

Tuxpeno core (landrace collection and populations) Veracruz 74

San Luis Potosi 57

Chiapas 50

Tamaulipas 23

Guatemala 22

Nayarit 20

Sinaloa 11

Hidalgo 7

Jalisco 7

Nuevo Leon 6

Other states and countries 44

GEM recommended lines (SS/NSS)* U.S. 54

CML: CIMMYT maize lines (A/B)* CIMMYT 94

U.S. landraces (Southern and Corn Belt Dent) U.S. 14

CIMMYT populations: breeding populations and single crosses CIMMYT 11

Temperate inbreds U.S. 3

China 1

Total 498

*GEM SS: stiff stalk synthetic heterotic group; GEM NSS: non-stiff stalk synthetic heterotic group; CML-A: CIMMYT maize line of heterotic group A; CML-B: CIMMYT maizeline of heterotic group B.doi:10.1371/journal.pone.0032626.t001



more weight than morphological distance because of more SNP

numbers than number of phenotypic traits (i.e., 1,433 vs. 44). The

resulting matrix D of combined distances showed to be an

Euclidean distance matrix as all the Eigen values from the

similarity matrix S = 12D were positive values, that is S was a

positive definite matrix.

Because the evaluation of phenotypic data was conducted in

seven different sets of trials, a sequential strategy was used to

obtain the mini core subset. First we defined the number of

accessions to be selected from each trial set according to the

diversity of each trial set. That is, the number of accessions we

selected is proportional to the average of distances between

accessions within each trial set:

ni~int 0:5z64|di

Sidi

� �

where ni is the number of accessions to be selected from the ith set,

di is the average of distances between accessions within the ith trial

set, and 64 is the number of accessions to be selected to form the

mini-core. Second, 1,000 mini-core subset candidates were

randomly and independently drawn following a stratified random

sample process of selection where each set was a stratum; then for

each candidate subset the average distance between its 64

accessions was calculated. Finally, the candidate showing maxi-

mum average distance between accessions was selected to be the

mini-core subset [38].

To evaluate the mini-core subset we used three concepts: (1) the

increase of the average of distances between accessions in the mini-

core in respect to the core set; (2) comparison of allele richness

(expected and observed heterozygosity); (3) comparison of means,

standard errors, and ranges between core and mini-core, and

calculus of the range recuperation (RR, %) in the mini-core. As

discussed by Marita et al. [39], allele richness is an evaluation from

the point of view of taxonomists or geneticists looking for core

subsets ensuring the inclusion of restricted or rare alleles; while

distances between accessions is an evaluation from the point of

view of breeders, looking for the inclusion of ‘‘generalized’’ alleles.

Results

Genotypic dataA total of 1,443 polymorphic SNPs (93.3%) were successfully

called, with less than 10% missing data in 350 accessions

(including 321 Tuxpeno core, 14 U.S. landraces, 11 CIMMYT

populations and 4 temperate inbreds, 647 plants in total). They

were evenly distributed across the whole maize genome, with

coverage ranging from 103 SNPs on chromosome 10 to 213 SNPs

on chromosome 1 (Table S3). Ninety-four CMLs and 54 GEM

lines were genotyped with a set of SNPs [40] that has 1,041

Figure 1. Neighbor-joining clustering of all 498 accessions based on the modified Rogers distance calculated using 1,041 SNPs.doi:10.1371/journal.pone.0032626.g001



markers in common with the 1,433 SNPs (Table S3). Marker

names and physical positions of these 1,433 SNPs are listed in

Table S3, where 1,041 out of 1,433 SNPs used for genotyping 148

GEM and CML lines were marked.

Dendrogram of all entriesThe Neighbor-joining tree of all 498 entries is shown in Fig. 1,

where lines from the same germplasm group (eg. Group of

Tuxpeno core, CMLs and GEM lines) tended to clustered

together. All U.S. landraces clustered together except one

accession named ‘‘Mexican June’’, which grouped with lines from

CIMMYT populations (La Posta-Across 8443, Population 23, 28,

32, and Pool 24). Entries from CIMMYT populations were

scattered next to the group of Tuxpeno core, except Population

21, which clustered amongst the Tuxpeno accessions. Pop 21 is

composed of seven Tuxpeno race accessions and some families

from Pool 24 (which is mainly based on Tuxpeno germplasm but

includes also some materials from Central America). Lines from

heterotic group SS and NSS of GEM were absolutely distin-

guished. Mo17 and the other three temperate inbred lines grouped

with GEM lines; Mo17 and CI7_1 were clustered in the NSS

group; K22_1 and DAN340 were clustered between NSS and SS

group. However, lines from heterotic groups A and B of CMLs

were not clearly separated. Grouping of different germplasm was

also shown in Fig. S1, where bootstrap value (%) above 50% was

shown. Tuxpeno accessions collected from the same region were

not necessarily grouped together (Fig. 2).

Genetic diversity among Tuxpeno core, GEM, CMLs andother germplasm

Gene diversity (expected heterozygosity) and observed hetero-

zygosity of different sets of germplasm revealed by SNP markers

are shown in Table 2. Using 1,433 SNPs, the set of U.S. landraces

have higher values for gene diversity and heterozygosity than

Tuxpeno core, temperate inbreds, and CIMMYT populations,

which may be due to the inclusion of Southern dent and Corn Belt

dent races in it [41]. The set of GEM lines has the highest values

for gene diversity among all the germplasm assembled in this

study, on the basis of 1,041 SNPs. This may result from the clear

heterotic groups (SS and NSS) within GEM lines ([26];http://

www.public.iastate.edu/,usda-gem/).

Genetic distances among Tuxpeno core, GEM-SS, GEM-NSS, CML-A and CML-B

Pair-wise MRD among Tuxpeno core, CML heterotic groups A

and B, GEM heterotic groups SS and NSS, as well as MRD within

each group are shown in Table 3. According to Tukey-Kramer

Figure 2. Neighbor-joining clustering of 321 Tuxpeno corebased on the modified Rogers distance calculated using 1,041SNPs.doi:10.1371/journal.pone.0032626.g002

Table 2. Genetic diversity of Tuxpeno core and other diverse germplasms studied by two sets of SNP markers.

Number of accessions Number of plants Gene diversity Heterozygosity

Tuxpeno core 321 618 0.2926 0.2558

Tuxpeno mini core 64 121 0.2986 0.2623

U.S. landraces 14 14 0.3078 0.3610

CIMMYT populations 11 11 0.2667 0.2724

Temperate inbreds 4 4 0.2745 0.0551

Tuxpeno core 321 618 0.2997 0.2607

Tuxpeno mini core 64 121 0.3056 0.2671

U.S. maize races 14 14 0.3235 0.3792

CIMMYT populations 11 11 0.2735 0.2768

Temperate inbreds 4 4 0.2859 0.0464

CML 94 94 0.2990 0.0110

GEM 54 54 0.3891 0.1217

doi:10.1371/journal.pone.0032626.t002



comparison of MRD means, larger genetic distances were

observed between Tuxpeno core and GEM groups than that

between Tuxpeno core and CML groups. MRD between CML

heterotic groups A and B were less than that between GEM

heterotic groups SS and NSS. The Tuxpeno core was closer to

GEM-NSS group than GEM-SS group, according to the genetic

distances. MRD within the Tuxpeno core was the least (Table 3).

Relationship among different germplasm groups based on MRD

was consistent with that based upon Nei’s genetic distance, as

revealed from Table 3 and Fig. S1.

Adaptation, genetic divergence and phenotypic variationof Tuxpeno core

The set of 321 Tuxpeno accessions represents 27 geographic

regions (Mexican states and other countries) of the landrace

adaptation, in which 10 major regions were identified. More than

5 accessions were collected from each of these 10 regions (Table 1).

In total, 299 out of 321 accessions were classified into their

corresponding MEs, based on available latitude, longitude and

altitude data. A total of 171 accessions from 16 states of Mexico

were classified as non-equatorial tropical/subtropical lowland wet

mega-environment (day length: 12.5 to 13.4 hours, mean

temperature $24uC, precipitation $600 mm and ,2000 mm).

The second largest group was classified into the tropical mid-

altitude mesic mega-environment (day length: 11 to 12.5 hours,

mean temperature .18uC and ,24uC, precipitation $200 mm

and ,600 mm), in which 41 Tuxpeno core accession from

Guatemala, and Chiapas, Tamaulipas, and Veracruz states in

Mexico were collected. Twenty-six Tuxpeno core accessions were

in non-equatorial tropical/subtropical lowland mesic (day length:

12.5 to 13.4 hours, mean temperature $24uC, precipitation

$200 mm and ,600 mm) and non-equatorial tropical/subtrop-

ical mid-altitude wet (day length: 12.5 to 13.4 hours, mean

temperature .18uC and ,24uC, precipitation $600 mm and

,2000 mm) mega-environments, respectively, which are the third

largest groups (Table S4).

The AMOVA (Table S5) revealed that a very low percentage

(1.30%) of variation was partitioned among the 10 subgroups of

Tuxpeno accessions. Only 9.74% of the variation was attributed to

differences among individuals within these 10 subgroups. The

majority of the variation was found within individuals (88.96%).

Pair-wise Fst among these 10 subgroups showed that in general the

accessions in Veracruz, Chiapas, and Guatemala were significantly

differentiated from those in most of other states in Mexico

(P#0.01). Accessions from Hidalgo showed no significant

differentiation as compared to those from all other subgroups

(Table 4). However, genetic differentiation based on molecular

data didn’t completely concur with the morphological Gower

distance (Table 5), suggesting no strong association between

molecular and phenotypic data in this study. Most accessions in

this Tuxpeno core are late white dent, with a few yellow late dent

accessions collected from Huasteca regions of Veracruz, Hidalgo,

and San Luis Potosi. CIMMYT populations have used most of

them, but perhaps much less have been exploited from Chiapas

and Guatemala.

The range and mean are summarized in Table 6 for certain

important agronomical and yield-related or reproductive traits of the

321 Tuxpeno accessions evaluated in the seven trial sets. Wide

variations were observed in days to 50% anthesis (AN), days to 50%

silking (SI), plant height (PH), ear height (EH), ear length (EL) and ear

diameter (ED). Other traits such as number of leaves above ear

(LAE), kernel length (KL), kernel width (KWD), and ratio of kernel

width to length (KWL) showed a relatively narrow range of variation.

Minicore subset of TuxpenoA minicore subset containing 64 accessions was defined. The

genetic diversity represented by gene diversity, heterozygosity and

Gower distance (Gd) in the minicore and core collections were

compared. Gene diversity and heterozygosity of the minicore

subset were higher than those of the core set (Table 2). In addition,

Table 3. Average and standard error of modified Rogers pair-wise genetic distances studied by 1,041 SNP markers within(diagonal) and between (lower diagonal) Tuxpeno core (Tux.core), CML heterotic groups, and GEM heterotic groups; number ofaccessions per group (n); results of the Tukey-Kramer comparison of group means (lower letters).

Group CML-A CML-B GEM-NSS GEM-SS Tux.core n

CML-A 0.56960.00082 e{ 48

CML-B 0.57060.00064 e 0.56160.00104 f 38

GEM-NSS 0.58560.00091 c 0.58760.00102 c 0.50260.00213 g 19

GEM-SS 0.65760.00064 a 0.65660.00072 a 0.63660.00104 b 0.58160.00112 d 35

Tux.core 0.48060.00022 i 0.47760.00025 j 0.49060.00035 h 0.57060.00026 e 0.33560.00011 k 321

{Means followed by the same letter indicated no difference in the Tukey-Kramer test.doi:10.1371/journal.pone.0032626.t003

Table 4. Pair-wise Fst studied based on 1433 SNPs for 10subgroups of Tuxpeno core classified according to the regionsthey were collected from (i.e., 9 states of Mexico andGuatemala).

CHIS GUAT HIDA JALI NAYA NVOL SINA SNLP TAMA VERA

CHIS 0

GUAT 0.015* 0

HIDA 0.012 0.022 0

JALI 0.014 0.029* 0.024 0

NAYA 0.014* 0.029* 0.021 0.013 0

NVOL 0.024* 0.040* 0.024 0.034 0.036 0

SINA 0.013* 0.028* 0.018 0.016 0.014* 0.023 0

SNLP 0.009* 0.022* 0.006 0.015 0.017* 0.018 0.011 0

TAMA 0.015* 0.029* 0.013 0.024 0.024* 0.011 0.013 0.009* 0

VERA 0.008* 0.013* 0.007 0.019* 0.018* 0.026* 0.017* 0.009* 0.014* 0

*Significant at the level P#0.01.CHIS = Chipas; GUAT = Guatemala; HIDA = Hildago; JALI = Jalisco;NAYA = Nayarit; NOVL = Nuevo Leon; SINA = Sinaloa; SNLP = San Luis Potosi;TAMA = Tamauripas; VERA = Veracruz.doi:10.1371/journal.pone.0032626.t004



Gd of the minicore subset (0.3289) was higher than that of the core

set (0.3159) as well. Finally the means, standard deviations and

ranges of 14 agronomical and yield related continuous variables

characterized for the entire core set were recovered in the

minicore (Table 6). Thus, the minicore subset reduced the number

of genotypes while maintaining the diversity of the core collection

(i.e. reducing the presence of some redundancies in the entire core

set), which is satisfactory. The collecting sites (states or depart-

ments in Mexico and Guatemala) and CIMMYT accession

identification numbers (Acc.ID) of these 64 Tuxpeno minicore

accessions are shown in Table S6.

Discussion

Genetic diversity of Tuxpeno core set and minicoresubset

The Tuxpeno core set for breeding use was chosen to best

represent phenotypic diversity within the race. They covered 23

States of Mexico, and parts of Brazil, Ecuador, Guatemala, and

Venezuela, including landraces and old breeding populations. A

relatively high gene diversity and heterogygosity were observed as

revealed by SNP markers. In addition, the geographic locations

(mega-environments) where the Tuxpeno core accessions were

collected show a wide climatic range. This confirmed a previous

study which indicated that Tuxpeno is the most widely adapted

Mexican landrace, as it is found in 19 climatic types [42].

Environmental differences seem to drive the overall patterns of

maize diversity [42,43]. Ecogeographical information where the

collections originated from is central to understanding the variety

of other sites in which they can adapt to. Breeders can select the

promising accessions with potential adaptation and use them in

the breeding program. The minicore subset, as indicated from the

present result, can capture the genetic variation present in the

Tuxpeno core set. We used a strategy combining phenotypic and

genotypic data to develop the minicore. A distance was defined

using both phenotypic and genotypic variables to achieve effective

classification of genotypes. Inclusion of morphological traits to

measure the distance is better than using only genotypic or marker

data, since they provided additional information generally

independent of the genotypic information. The use of the weighted

average of both morphological and genetic distance followed the

Gower principle, in which more variables produce larger effects.

Evaluation of agronomically important and stress-tolerant traits

can be carried out using the minicore. Mining new alleles for

useful traits either in the minicore or in the core is cost-effective, as

the number of accessions is substantially reduced compared to that

of the entire Tuxpeno race collection at the CIMMYT maize

germplasm bank.

The present study on the core set of the largest collection in

CIMMYT (i.e. race Tuxpeno) can be extended and applied to

other landrace collections. As shown in Figure 2, relationship

among the accessions does not necessarily follow the geographic

pattern for the collection of the accessions. Hence, genotyping a

large number of accessions and plants per accession would be

necessary in order to establish relationship among the landraces

and devise sampling strategy in the future.

Grouping of GermplasmClustering analysis based on MRD and Nei’s genetic distance

revealed clear separation among different germplasm (Fig. 1; Fig.

Table 5. Average of Gower pair-wise phenotypic distances within (diagonal) and between (lower diagonal) 10 subgroups ofTuxpeno core originated from 9 states of Mexico and Guatemala; standard errors of the means (in parenthesis); results of theTukey-Kramer comparison of means (lower letters); number of accessions in each subgroup (n).

CHIS GUAT HIDA JALI NAYA NVOL SINA SNLP TAMA VERA n

CHIS 0.180 dc{ 50

(0.0019)

GUAT 0.189 bdc 0.182 bdc 22

(0.002) (0.0044)

HIDA 0.204 bac 0.212 bac 0.22 bac 7

(0.0037) (0.0057) (0.0158)

JALI 0.186 bdc 0.191 bdc 0.204 bac 0.199 bdac 7

(0.0036) (0.0055) (0.01) (0.0152)

NAYA 0.212 bac 0.217 bac 0.203 bac 0.217 bac 0.201 bac 20

(0.0022) (0.0034) (0.0059) (0.0061) (0.005)

NVOL 0.205 bac 0.215 bac 0.198 bdac 0.211 bac 0.185 bdc 0.191 bdac 6

(0.004) (0.0062) (0.0107) (0.011) (0.0061) (0.0177)

SINA 0.214 bac 0.224 a 0.207 bac 0.224 ba 0.197 bdac 0.182 bdc 0.199 bdac 11

(0.003) (0.0046) (0.008) (0.0083) (0.0046) (0.0082) (0.0093)

SNLP 0.195 bdac 0.203 bac 0.205 bac 0.205 bac 0.204 bac 0.194 bdac 0.203 bac 0.199 bac 57

(0.0012) (0.0019) (0.0035) (0.0035) (0.002) (0.0037) (0.0028) (0.0017)

TAMA 0.169 dc 0.181 dc 0.186 bdc 0.178 dc 0.189 bdc 0.176 dc 0.188 bdc 0.176 dc 0.147 d 23

(0.0019) (0.0029) (0.0053) (0.0052) (0.0031) (0.0056) (0.0042) (0.0018) (0.0038)

VERA 0.211 bac 0.214 bac 0.211 bac 0.219 bac 0.200 bac 0.195 bdac 0.206 bac 0.206 bac 0.194 bdac 0.199 bdac 74

(0.0011) (0.0017) (0.0031) (0.0031) (0.0018) (0.0032) (0.0024) (0.001) (0.0016) (0.0013)

{Means followed by the same letter indicated no difference in the Tukey-Kramer test.doi:10.1371/journal.pone.0032626.t005



S1). No subclusters were formed within the Tuxpeno core, which

is consistent with a high within individual variation (89%) revealed

by AMOVA (Fig. 2, Table S5). A total of 94 CMLs were not well

separated into A (mostly dent type) or B (flint type) patterns, as

conventional heterotic groups classified by the CIMMYT

breeders. This is as expected because most germplasm sources

used to extract the lines were established based on a mixture of

different racial complexes [44,45]. Similar results were demon-

strated in previous studies [46,47]. For CMLs analyzed in this

study, more than 50% of their base populations included Tuxpeno

germplasm (dent kernel) in their formation as CIMMYT gene

pools and populations used Tuxpeno germplasm for its high

productivity per se and good combination with other germplasm

(Table S1; [13]). This can be reflected by the relatively low genetic

distance between the CMLs and Tuxpeno core (Table 3).

On the other hand, 54 U.S. GEM recommended lines showed

two clear groups of NSS and SS heterotic patterns. The Tuxpeno

core had the largest genetic distance from GEM-SS lines among its

genetic distances from all other groups. In this study, larger genetic

distance between tropical germplasm (i.e. Tuxpeno core, CML-A

and CML-B) and SS were observed than that between tropical

germplasm and NSS, which is consistent with a previous study

[48]. A large genetic distance between heterotic germplasm can be

useful for developing lines with good combining ability in hybrid

breeding [49,50]. GEM-SS can be an excellent heterotic

germplasm against CML-A, CML-B and Tuxpeno germplsms,

considering these CMLs analyzed in this study did not show large

MRD from the other germplasm groups.

The gene diversity parameter used for evaluating the genetic

diversity in this study is less sensitive to the sample sizes of the

subsets [11,51]. However, the allele number of each locus is

restricted to a maximum of two when using bi-allelic SNP

markers, which may cause limitations in genetic diversity

measurement. Detection of genetic diversity with a large number

of SNPs could mitigate the shortage. In addition, ascertainment

biases might affect the measurement of diversity and population

differentiation due to the use of SNP genotyping chips. The

frequency of alleles may be affected and difference among

temperate lines may be overestimated compared to that within

tropical lines, because most SNPs (1106 out of 1536) used in the

present study were developed from sequencing the set of 27

parental lines of the nested association mapping (NAM) population

(i.e., SNPs were selected to maximize polymorphisms between B73

and 26 other inbred parental genotypes. About half of the 26 lines

are tropical.) [30]. With the availability of maize genome and the

advance of genotyping by sequencing technology, larger amount

SNPs with good quality can be used for molecular characterization

of maize landraces, which is possible to control ascertainment bias

[52,53,54].

Further use of Tuxpeno core set in maize breedingprograms

Tuxpeno germplasm has been exploited in tropical maize

improvement for its yield potential [55–57], superior plant type

[58,59], and resistance to drought and pests [60,61]. They

constitute the largest collection in the CIMMYT maize germplasm

bank. Despite much larger genetic distances and allelic frequency

differences between Tuxpeno and GEM groups than that between

Tuxpeno and CML groups, the results of cluster analysis showed

clear separation of CMLs from Tuxpeno. The divergence between

them implies that there may be untapped allelic variations in

Tuxpeno germplasm, which can be used for broadening the

genetic diversity within CML-A or B groups.

The 54 GEM lines investigated in our study have a 50% or 75%

background of temperate germplasm and a 25% or 50%

background of tropical germplasm. The genetic diversity of

GEM was broader in this study, compared to the tropical

germplasm (i.e. CML and Tuxpeno). However, large allelic

frequency differences between GEM and tropical germplasm

imply that the tropical germplasm can be used in a temperate

breeding program. Incorporation of elite tropical and subtropical

germplasm into elite temperate germplasm to combine favorable

alleles into germplasm pools adapted to temperate environments

as well as to broaden its genetic base have been carried out in

previous studies [62,63]. Whitehead et al. [62] suggested that 25%

elite exotic germplasm can be incorporated in the important U.S.

heterotic groups without disrupting the highly productive

combining ability for grain yield expressed in BSSS and non-

BSSS hybrid combinations. On the other hand, GEM germplasm

can be considered as an exotic source for improving tropical maize

lines and populations. Promising results were observed in the

breeding crosses, where clearer separation was observed between

the F1 crosses from CML A6GEM-SS and CML B6GEM-NSS

[40].

Larger separation between GEM heterotic groups (i.e. SS and

NSS), compared to the genetic divergence between CML heterotic

groups (i.e. CML-A and CML-B) provide tropical and temperate

maize breeders with potential germplasm sources for hybrid maize

breeding, in which the genetic distances between opposite

heterotic lines and populations can be increased. For example,

we can make allied breeding cross combinations between GEM-

SS and CML-A (or Tuxpeno minicore), and between GEM-NSS

and CML-B (or Tuxpeno minicore). GEM lines are subtropical-

temperate adapted and more tropical germplasm should be

Table 6. Statistical description of 14 agronomical and yieldrelated traits of Tuxpeno core and selected mini-coreevaluated from seven trials at CIMMYT stations.

------- core (321) ------- ----- mini-core (64) -----

Trait{ MeanStdDev Range Mean

StdDev Range RR1 %

AN (Day) 84.3 11.7 48.1 84.3 12.4 48.1 100.0

SI (Day) 86.6 12.0 54.1 86.6 13.0 54.1 100.0

PH (cm) 270.4 25.8 149.6 270.4 27.8 103.9 69.5

EH (cm) 172.7 23.9 143.4 172.7 27.6 122.1 85.1

LAE (No.) 6.3 0.5 2.3 6.3 0.5 2.3 99.2

EL (cm) 16.8 1.2 13.3 16.8 1.3 7.8 58.1

ED (cm) 4.7 0.3 3.2 4.7 0.3 1.7 53.1

KL (cm) 1.16 0.08 0.48 1.16 0.09 0.44 91.9

KWD (cm) 0.94 0.04 0.46 0.94 0.05 0.36 78.3

KRN (No.) 13.0 1.1 6.6 12.73 1.28 5.6 85.1

EDL (Ratio) 0.28 0.02 0.14 0.28 0.02 0.12 87.9

COB (cm) 2.42 0.31 3.17 2.42 0.30 1.42 44.7

CED (Ratio) 0.51 0.05 0.51 0.51 0.04 0.21 40.3

KWL (Ratio) 0.82 0.06 0.39 0.82 0.06 0.25 64.3

1Percentage of the range in the entire core recovered by the minicore subset.{AN = days to 50% anthesis; SI = days to 50% silking; PH = plant height; EH = earheight; LAE = number of leaves above the ear; EL = ear length; ED = eardiameter; KL = kernel length; KWD = kernel width; KRN = kernel row number;EDL = ratio of ear diameter to ear length; COB = cob diameter; CED = ratio of cobdiameter to ear diameter; KWL = ratio of kernel width to kernel length.doi:10.1371/journal.pone.0032626.t006



incorporated for its use in tropical breeding. In the above breeding

cross combinations, selection for tropically adapted SS-A heterotic

pattern and NSS-B heterotic pattern is recommended for tropical

maize breeding. Although Tuxpeno is one of the heterotic patterns

in tropical maize breeding, it may contribute to enhancing GEM-

SS heterotic lines. The same can be done with Tuxpeno minicore

for enhancing CML-A and CML A/B in the similar grain types.

Selection for adaptation and increasing genetic divergence must be

done as a priority using standard breeding procedures. As a result,

superior lines and hybrids can be developed in the adapted

regions.

In addition, short stature improved populations and lines of

Tuxpeno germplasm are good sources for improving the farmers’

landraces, without altering grain type and adaptation. CIMMYT

maize genebank has used the improved gene pools and lines in

participatory maize breeding in the state of Oaxaca, Mexico (Taba

et al. unpublished data; [20]) for evolutional maize germplasm

conservation. In this way, genetic diversity of the race can be

maintained in situ on farm [64] and modern maize production can

be realized with small scale farmers.

Supporting Information

Figure S1 Dendrogram of different germplasm groups (Tux-

peno core, CML-A, CML-B, CML-A/B, GEM-SS, GEM-NSS,

CIMMYT populations, U.S. landraces). Clades with greater than

50% bootstrap support are indicated.

(PPT)

Table S1 Information of lines collected and characterized in this

study.

(XLS)

Table S2 Detailed information of 321 Tuxpeno accessions.

(XLS)

Table S3 Information of 1,433 SNPs used in this study.

(XLS)

Table S4 Distribution of Tuxpeno core accessions (299

accessions with available information) by the collection informa-

tion in maize mega-environments (MEs) defined by a GIS

approach.

(DOC)

Table S5 Analysis of molecular variance of 10 subgroups of

Tuxpeno accessions classified according to the 10 major

geographic regions where they were collected.

(DOC)

Table S6 Collecting sites (states or departments in Mexico and

Guatemala) and CIMMYT accession identification number

(Acc.ID) of 64 Tuxpeno minicore accessions.

(DOC)

Acknowledgments

We are grateful to members of the maize germplasm bank at CIMMYT for

the field evaluations conducted at CIMMYT stations. Thanks also to the

U.S.-GEM project for their GEM lines, and assistance from CIMMYT

with development of core subsets of the CIMMYT maize collection.

Author Contributions

Conceived and designed the experiments: WW ST JY. Performed the

experiments: WW VHC JY ST. Analyzed the data: WW JF VHC JY.

Contributed reagents/materials/analysis tools: ST JY. Wrote the paper:

WW ST JF.

References

1. McClintock B, Kato-YTA, Blumenschein A (1981) Chromosome constitution of

races of maize. Colegio de postgraduados, Chapingo, Mexico.

2. Buckler ES, Phelps-Durr T, Buckler CK, Dawe AK, Doebley J, et al. (1999)

Meiotic drive of chromosomal knobs reshaped the maize genome. Genetics 153:

415–426.

3. Doebley J, Goodman M, Stuber CW (1985) Isoenzyme variation in the races of

maize from Mexico. American Journal of Botany 72: 629–639.

4. Doebley JF, Goodman M, Stuber CW (1986) Exceptional divergence of

northern flint corn. American Journal of Botany 73: 64–69.

5. Bretting PK, Goodman M, Stuber CW (1990) Isozymatic variation in

Guatemalan races of maize. American Journal of Botany 77: 211–225.

6. Sanchez GJJ, Stuber CW, Goodman M (2000) Isozymatic diversity in the races

of maize of the Americas. Maydica 45: 185–203.

7. Sanchez GJJ, Goodman M, Stuber CW (2000) Isozymatic and morphological

diversity in the races of maize of Mexico. Economic Botany 54: 43–59.

8. Sanchez GJJ, Goodman M, Bird RM, Stuber CW (2006) Isozyme and

morphological variation in maize of five Andean countries. Maydica 51: 25–42.

9. Matsuoka Y, Vigouroux Y, Goodman M, Sanchez J, Buckler E, et al. (2002) A

single domestication for maize shown by multilocus microsatellite genotyping.

Proceedings of the National Academy of Sciences, USA 99: 6080–6084.

10. Reif JC, Warburton ML, Xia XC, Hoisington DA, Crossa J, et al. (2006)

Grouping of accessions of Mexican races of maize revisited with SSR markers.

Theoretical and Applied Genetics 113: 177–185.

11. Vigouroux Y, Glaubitz JC, Matsuoka Y, Goodman MM, Sanchez GJ, et al.

(2008) Population structure and genetic diversity of new world maize races

assessed by DNA microsatellites. American Journal of Botany 95(10):

1240–1253.

12. Sharma L, Prasanna BM, Ramesh B (2010) Analysis of phenotypic and

microsatellite-based diversity of maize landraces in India, especially from the

North East Himalayan region. Genetica 138: 619–631.

13. CIMMYT (1998) A complete listing of improved maize germplasm from

CIMMYT. Maize Program Special Report. Mexico, D.F.

14. Vasal SK, Srinivasan G, Beck DL, Crossa J, Pandey S, et al. (1992) Heterosis

and combining ability of CIMMYT’s tropical late white maize germplasm.

Maydica 37: 217–223.

15. Wellhausen EJ (1978) Recent developments in corn breeding in tropics. In:

Walden DB, ed. Corn breeding and genetics, Wiley, New York. pp 59–84.

16. Bjarnason M, Pixley K (1994) Evaluation of Tuxpeno accessions from

subtropical areas. In: The CIMMYT Maize Germplasm Bank: Genetic

Resource Preservation, Regeneration, Maintenance, and Use. Maize Program

Special Report. Mexico, D.F.

17. Franco J, Crossa J, Villasenor J, Taba S, Eberhart SA (1998) Classifing genetic

resources by categorical and continuous variables. Crop Sci 38: 1688–1696.

18. Franco J, Crossa J, Taba S, Shands H (2005) A sampling strategy for conserving

genetic diversity when forming core subsets. Crop Sci 45: 1035–1044.

19. Seron-Rojas JJ, Crossa J, Sahun-Castellanos J, Castillo-Gonzalez F, Santacruz-

Varela A (2006) A selection index method based on eigenanalysis. Crop Sci 46:

1711–1721.

20. Ortiz R, Taba S, Tovar VHC, Mezzalama M, Xu Y, et al. (2010) Conserving

and enhancing maize genetic resources as global public goods–A perspective

from CIMMYT. Crop Sci 50: 1–16.

21. Taba S, Diaz J, Rivas M, Rodriguez M, Vicarte V, et al. (2003) The CIMMYT

maize collection: preliminary evaluation of accessions. In: 7762 unique

accessions from Mexico, the Caribbean and Central America and some

countries of South America. CD-ROM.

22. Taba S (1995) Maize germplasm: Its Spread, Use, and Strategies for

Conservation. 7–58. in Taba S, ed. 1995. Maize Genetic resources. Maize

program special report. Mexico D.F.: CIMMYT.

23. Taba S (1997) Maize. 213–226. In Biodiversity in Trust, conservation and use of

plant genetic resources in CGIAR Centers Fuccillo D, Sears L, Stapleton P, eds.

Cambridge University Press, Cambridge, UK.

24. Gower JC (1971) A general coefficient of similarity and some of its function

properties. Biometrics 27: 857–874.

25. Salhuana W, Sevilla R, eds (1995) Latin American Maize Project (LAMP), stage

4 results from homologous areas 1 and 5 (Catalog and CD-ROM). National

Seed Storage Laboratory, Fort Collins, CO.

26. Salhuana W, Pollak LM, Ferrer M, Paratori O, Vivo G (1998) Agronomic

evaluation of maize accessions from Argentina, Chile, the United States, and

Uruguay. Crop Sci 38: 866–872.

27. Pollak LM (2002) The history and success of the public-private project on

germplasm enhancement of maize (GEM). Advances in Agronomy 78: 45–87.

28. Salhuana W, Pollak LM (2006) Latin American Maize Project (LAMP) and

Germplasm Enhancement of Maize (GEM) Project: Generating useful breeding

germplasm. Maydica 51: 339–355.

29. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight

plant DNA. Nucleic Acids Res 8: 4321–4325.

30. Yan JB, Yang XH, Hector S, Sanchez H, Li JS, et al. (2010) High-throughput

SNP genotyping with the GoldenGate assay in maize. Mol Breed 25: 441–451.



31. Fan JB, Gunderson KL, Bibikova M, Yeakley JM, Chen J, et al. (2006) Illumina

universal bead arrays. Methods Enzymol 410: 57–73.32. Nei M (1972) Genetic distance between populations. Am Nat 106: 283–292.

33. Nei M (1987) Molecular evolutionary genetics. Columbia Univ. Press, New

York.34. Hartkamp AD, White JW, Aguilar AR, Banziger M, Srinivasan G, et al. (2000)

Maize production environments revisited: A GIS-based Approach [Online].CIMMYT website. Available: http://www.cimmyt.org/Research/NRG/map/

research_results/nrg_papers/GIS_maprod/MaProd_cont.htm. Accessed 2005

April 15).35. Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: an integrated

software package for population genetics data analysis. Evol Bioinform Online 1:47–50.

36. Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular varianceinferred from metric distances among DNA haplotypes: application to human

mitochondrial DNA restriction data. Genetics 131: 479–491.

37. Reif JC, Melchinger AE, Frisch M (2005) Genetical and mathematicalproperties of similarity and dissimilarity coefficients applied to plant breeding

and seed bank management. Crop Sci 45: 1–7.38. Franco J, Crossa J, Deshande S (2010) Hierarchical multiple-factor analysis for

classifying genotypes based on phenotypic and genetic data. Crop Sci 50:

105–117.39. Marita JM, Rodriguez JM, Niehuis J (2000) Development of an algorithm

identifying maximally diverse core collections. Genet Resour Crop Evol 47:515–526.

40. Wen WW, Guo TT, Chavez Tovar VH, Li HH, Yan JB, et al. (2012) Thestrategy and potential utilization of temperate germplasm for tropical germplasm

improvement: a case study of maize (Zea mays L.). Mol Breeding. DOI 10.1007/

s11032-011-9696-1.41. Goodman MM, Brown WL (1988) Races of corn. In: Corn and corn

improvement. Third edition Sprague GF, Dudley JW, eds. ASA, CSSA, SSSApublishers, Madison, Wisconsin, USA.

42. Corral JAR, Puga ND, Sanchez J, Parra JR, Eguiarte DRG, et al. (2008)

Climatic Adaptation and Ecological Descriptors of 42 Mexican Maize Races.Crop Sci 48: 1502–1512.

43. Brush SB, Perales HR (2007) A maize landscape: Ethnicity and agro-biodiversityin Chiapas, Mexico. Agric Ecosyst Environ 121: 211–221.

44. Vasal SK, Cordova H, Pandey S, Srinivasan G (1999) Tropical maize andheterosis. In: Coors JG, Pandey S, eds. The genetics and exploitation of heterosis

in crops, ASA, CSSA and SSSA, Madison, WI. pp 363–373.

45. Reif JC, Melchinger AE, Xia XC, Warburton ML, Hoisington DA, et al. (2003)Genetic distance based on simple sequence repeats and heterosis in tropical

maize populations. Crop Sci 43: 1275–1282.46. Xia XC, Reif JC, Hoisington DA, Melchinger AE, Frisch M, et al. (2004)

Genetic diversity among CIMMYT maize inbred lines investigated with SSR

markers: I. Lowland tropical maize. Crop Sci 44: 2230–2237.47. Xia XC, Reif JC, Melchinger AE, Frisch M, Hoisington DA, et al. (2005)

Genetic diversity among CIMMYT maize inbred lines investigated with SSR

markers: II. Subtropical, tropical midaltitude, and highland maize inbred lines

and their relationships with elite U.S. and European maize. Crop Sci 45:2573–2582.

48. Liu KJ, Goodman M, Spencer M, Smith JS, Buckler ES, et al. (2003) Genetic

structure and diversity among maize inbred lines as inferred from DNAmicrosatellites. Genetics 165: 2117–2128.

49. Melchinger AE (1999) Genetic diversity and heterosis. In: JG. Coors, S.Pandey, eds. The genetics and exploitation of heterosis in crops, ASA, CSSA,

and SSSA, Madison, WI. pp 99–118.

50. Moll RH, Longquist JH, Fortuna JV, Johnson EC (1965) The relation ofheterosis and genetic divergence in maize. Genetics 52: 139–144.

51. Petit RJ, Mousadik AE, Pons O (1998) Identifying populations for conservationon the basis of genetic markers. Conservation Biology 12: 844–855.

52. Gore MA, Chia JM, Elshire RJ, Sun Q, Ersoz ES, et al. (2009) A first-generationhaplotype map of maize. Science 326(5956): 1115–1117.

53. Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, et al. (2009) The B73 maize

genome: complexity, diversity, and dynamics. Science 326: 1112–1115.54. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, et al. (2011) A robust,

simple genotyping-by-sequencing (GBS) approach for high diversity species.PLos One 6(5): e19379.

55. Pandey S, Vasal SK, Deutsch JA (1991) Performance of open-polinated maize

cultivars selected from 10 tropical maize populations. Crop Sci 31: 285–290.56. Vasal SK, Beck DL, Crossa J (1986) Studies on the combining ability of

CIMMYT maize germplsam. In: CIMMYT Research highlights, Mexico D.F.:CIMMYT. pp 24–33.

57. Han GC, Vasal SK, Beck DL, Elias E (1991) Combining ability of inbred linesderived from CIMMYT maize (Zea mays L) germplasm. Maydica 36: 57–64.

58. Johnson EC, Fischer KS, Edmeades GO, Palmer AFE (1986) Recurrent

selection for reduced plant height in lowland tropical maize. Crop Sci 26:253–260.

59. Fischer KS, Edmeades GO, Johnson EC (1987) Recurrent selection for reducedtassel branch number and reduced leaf area density above the ear in tropical

maize populations. Crop Sci 27: 1150–1156.

60. Fischer KS, Edmeades GO, Johnson EC (1989) Selection for the improvementof maize yield under moisture-deficits. Field Crops Res 22: 227–243.

61. Smith ME, Mihm JA, Gracen VE (1983) Genetics of the reaction to fallarmyworm in Tuxpeno and Antigua maize populations. In: Agronomy Abstracts

1983, Madison, Wisconsin: American Society of Agronomy. 81 p.62. Whitehead FC, Caton HG, Hallauer AR, Vasal S, Cordova H (2006)

Incorporation of elite subtropical and tropical maize germplasm into elite

temperate germplasm. Maydica 51: 43–56.63. Mungoma C, Pollak LM (1988) Heterotic patterns among ten Corn Belt and

exotic maize populations. Crop Sci 28: 500–504.64. Duvick D (1993) Possible effects of intellectual property rights on erosion and

conservation of plant genetic resources in centers of crop diversity. In:

Buxton DR, Shibles R, Forsberg RA, Blad BL, Asay KH, Paulsen GM,Wilson RF, eds. International Crop Sci I, CSSA, Madison, Wisconsin, USA. pp

505–509.



Genetic Characterization of a Core Set of a Tropical Maize Race Tuxpeño for Further Use in Maize Improvement

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